Chromatographic-mass Spectrometric Technique Research Articles

Simultaneous determination of sorbic acid, benzoic acid and parabens in foods: a new gas chromatography-mass spectrometry technique adopted in a survey on Italian foods and beverages.

A gas chromatographic-mass spectrometric technique is described for the simultaneous determination of sorbates, benzoates and other lipophilic preservatives in foods and beverages. The selected ions monitoring (SIM) technique allowed unambiguous identification of the compounds under study. This methodology eliminated all kinds of interferences from the complex food matrices which affect most routinely-used techniques, HPLC included. A very simple and time-saving extraction procedure was therefore employed, since subsequent purification steps were unnecessary, even for detection of trace levels of preservatives. The detection limits fell within the range of 100-200 pg. With this analytical technique, we have conducted a survey on sorbic acid, benzoic acid, methyl, ethyl, and propyl 4-hydroxybenzoate levels in 249 samples of foods and beverages on sale in markets in the Rome area. Samples were chosen from among those currently preserved by these additives. All compounds were also determined by a routinely-used HPLC technique for method comparison.

Thermal Desorption of Organic Compounds Associated with Settled Household Dust

Settled household dust samples were collected from twelve dwellings in urban areas during an annual winter heating period. Emission of compounds from settled household dust was analyzed under simulated hot surface conditions with a temperature range of 50–300°C. The compounds were analyzed and identified by thermal desorption—gas chromatographic-mass spectrometric technique. The organic emission from household dust was relatively low at temperatures below 70° C, increased appreciably above 100°C, and gained in strength at temperatures above 200°C. Desorption of adsorbed compounds is the main contribution to emissions, but at higher temperatures the thermal degradation seems to affect also the quantity and the quality of the emissions. The organic composition of household dust was found to be equal in quality at different sampling sites; the emissions consist of mainly aliphatic aldehydes (C6—C13), aliphatic carboxylic acids and their esters (C8 C18, C6–C30) and phthalates. Phosphate esters, branched alkanes, n-alkenes, n-alkanones, monoterpenes, aromatic hydrocarbons, and aromatic and aliphatic alcohols were also well represented groups in household dust samples. The potential sources of identified compounds are discussed.

Analysis of polychlorinated dibenzofurans (PCDFs) in fly ash from a waste incineration plant.

In this study, a method for the congener-specific analysis of polychlorinated dibenzofurans (PCDFs) in fly ash from a waste incineration plant is described. This method involves extraction, column chromatography, and gas chromatographic-mass spectrometric techniques. Representative fly ash samples are obtained from the waste incinerator. The purpose of this research is to briefly present the findings as they relate to PCDFs. Analyses indicate that PCDFs are consistently found in the municipal incinerator.

Sequence distribution of styrene-butadiene copolymers by ozonolysis, high-performance liquid chromatographic and gas chromatographic-mass spectrometric techniques

The sequence distribution of styrene units in various styrene-butadiene copolymers (SBR) was investigated by liquid chromatographic (LC) and gas chromatographic-mass spectrometric (GC-MS) measurements on their ozonolysis products. Ozonolysis was performed in methylene chloride followed by reductive degradation with lithium aluminium hydride. LC was found to be a very effective technique for the characterization and quantitative evaluation of the sequence distribution of random and tapered block copolymers because of its high detection efficiency of short and long sequences. LiChrospher C 18 reversed-phase columns, a ternary gradient system and an evaporative light-scattering detector were used. Peaks corresponding to various low-molecular-mass species were identified by GC-MS and assigned to 1,4Bde-(Sty) m -1,4Bde or 1,4Bde-(Sty) m -(1,2Bde) n -1,4Bde sequences of the original copolymers (Bde = butadiene, Sty = styrene).

Lithologic Variation of the Rare Biomarker Compound 28, 30-Bisnorhopane Within the Miocene Monterey Formation in the Texaco Anita-14 Well, Santa Barbara County, California: ABSTRACT

The rare geological marker compound 28, 30-bisnorhopane is commonly found in the oils and rocks of the Miocene Monterey Formation of central and southern California. In the offshore Texaco Anita-14 well of the Santa Barbara coastal area, the most significant concentrations of 28, 30-bisnorhopane are restricted to a small interval of organic-rich, calcareous/phosphatic shale. Onshore exposures of the Monterey Formation in this area are commonly subdivided into five lithologic members, all of which have been identified in the Anita-14 well based on visual examination and XRD analysis of the core and cuttings coupled with electric log interpretations. Soluble organic matter was extracted from several core and cuttings samples from each lithologic member and analyzed for hopanes by standard gas chromatographic-mass spectrometric (GC-MS) techniques. Preliminary results reveal minor, but somewhat variable concentrations of 28, 30-bisnorhopane in all of the Monterey members but one. The greatest concentration of 28, 30-bisnorhopane occurs within a small interval of poorly laminated, organic-rich calcareous/phosphatic shale that also contains the highest amount of extractable organic matter in the core. Furthermore, within this organic-rich member, bisnorhopane concentrations appear to be higher in the more strongly laminated rocks as opposed to those that are weakly laminated or massive. Thismore » evidence supports the proposition that 28, 30-bisnorhopane may be of use as an indicator of depositional conditions for one of the more organic-rich source rocks of the Monterey Formation.« less

Diagnosis and monitoring of disseminated candidiasis based on serum/urine D/L-arabinitol ratios

Disseminated candidiasis, a devastating disease with high morbidity and mortality in immunosuppressed patients, is difficult to diagnose because of the protean nature of symptoms and the lack of rapid and reliable laboratory diagnostic procedures. The subject of this review is the status of gas chromatographic-mass spectrometric techniques for the determination of D-arabinitol, a unique metabolite of pathogenic Candida species, in serum and urine. The enantiomers are separated by chiral chromatography followed by specific and sensitive detection using chemical ionization and selected ion monitoring. Using D/L-arabinitol ratios, instead of individual concentrations, eliminates the need for knowing the volume of samples and for calibration curves. A new filter paper technique requires only an unmeasured drop of whole blood (venous or finger/heel puncture) or urine; paper spots are mailable. Parallel determinations of D/L-arabinitol ratios in serum and urine in normal subjects and cancer patients with both normal and increased D/L-arabinitol ratios revealed constant (1.2-1.3 range) ratios of serum D/L-arabinitol/urine D/L-arabinitol for all populations studied. Analyzing two body fluids taken at the same time increases reliability by reducing false positives.

Widespread distribution of free d-aspartate in rat periphery

We have identified and quantified free d-aspartate in adult rat peripheral organs using gas chromatographic-mass spectrometric and high-performance liquid Chromatographic techniques. The level of free d-aspartate was highest in the adrenal, testis, spleen and pituitary, followed by the thymus, lung, ovary, placenta, pancreas and heart, and below the detection limit in the kidney, liver, brain, muscle and serum. These data provide the first evidence that a high level of free d-aspartate widely occurs in the adult rat periphery and suggest that the d-amino acid may be an endogenous substrate for d-aspartate oxidase.

Development of a high-performance liquid chromatographic—mass spectrometric technique, with an ionspray interface, for the determination of platelet-activating factor (PAF) and lyso-PAF in biological samples

An HPLC-mass spectrometric technique with an ionspray interface was developed for the determination of platelet-activating factor (PAF) and PAF-related compounds in biological samples. HPLC separations were performed using a reversed-phase column. The mass spectra showed intense [M + H] + ions. Collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the phosphorylcholine group. By selective-ion monitoring, a detection limit of 0.3 ng was obtained for all molecules; by multiple reaction monitoring, the same sensitivity was achieved for PAF whereas for lyso-PAF the limit was 3 ng. Finally, PAF was comparatively determined by bioassay and HPLC-MS after extraction from the cell pellets and the supernatants of human polymorphonuclear neutrophils unstimulated or stimulated with opsonized zymosan. The good correlation observed between these techniques indicated the reliability of HPLC-MS for biochemical studies on PAF and PAF-related molecules.

Biotransformation of diethenylbenzenes: IV. A simple high-performance liquid chromatographic method for separation of urinary metabolites of 1,3-diethenylbenzene on analytical and semi-preparative scales

A simple ion-suppression separation on reversed-phase columns, which is applicable for both analytical and semi-preparative work, is described. Six urinary metabolites of 1,3-diethenylbenzene (I), namely 1-(3-ethenylphenyl)-1,2-dihydroxyethane β- d-glucosiduronates (two isomers, II and III), N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]cysteine (IV), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]cysteine (V), 3-ethenylphenylmandelic acid (VI) and 3-ethenylphenylglyoxylic acid (VII), were isolated (Fig. 1). Four of them, IV–VII, have been identified in our previous work; the two glucosiduronates were identified for the first time by 1H NMR spectroscopy, fast atom bombardment mass spectrometry, and enzymic hydrolysis yielding 1-(3-ethenylphenyl)-1,2-dihydroxyethane as an aglycone. The method was reproducible the concentration range 0.05–5 mg/ml, the coefficient of variation being less than 7% ( n = 5). Excretion of II–VI within 24 h in the urine of rats dosed with a single intraperitoneal injection of 100, 300 and 600 mg/kg I was determined quantitatively. The utility of the method is discussed in comparison with gas chromatographic—mass spectrometric techniques used previously.

High-performance liquid chromatographic-mass spectrometric analysis of oligosaccharides from enzymatic digestion of glycosaminoglycans: Application to human samples

Glycosaminoglycan contents were evaluated in plasma and urine samples from volunteers treated intravenously with a mixture of dermatan sulphate and heparin, combining a novel liquid chromatographic-mass spectrometric technique for the determination of oligosaccharides from glycosaminoglycans with a classical technique for the extraction of glycosaminoglycans from biological samples (precipitation with cetylpyridinium chloride). In plasma samples dermatan sulphate and heparin can be measured for 2 h after treatment; urine excretion was detectable for 24 h. These results suggest that this novel approach is promising for future studies on the pharmacokinetics of glycosaminoglycans, although some technical aspects need further improvement, mainly regarding the procedures for sample clean-up; cetylpiridinium precipitations is a complex procedure and the recovery is limited.

Cell Wall Metabolism in Ripening Fruit

A gas chromatographic-mass spectrometric technique utilizing d-[U-(13)C]glucose as a density label tracer was used to follow the synthesis of cell wall polysaccharides in pericarp discs that were excised from mature green tomato fruit (Lycopersicon esculentum) and allowed to ripen in culture. The biosynthetic capacity of discs from four different maturity stages was examined. Label was differentially incorporated into wall polysaccharides as the discs matured, indicating a change in the nature of wall polymers being synthesized. These differential rates of incorporation are consistent with descriptions of ripening-related cell wall compositional changes previously reported by other authors. Specific changes in wall biosynthesis noted include increased incorporation of xylosyl and mannosyl residues into hemicellulosic cell wall fractions as the discs mature and decreased incorporation of galactosyl residues into chelator-soluble pectins.

A gas chromatographic-mass spectrometric technique for studying simultaneous hydrogen-deuteron exchange and para-orthohydrogen conversion in hydrogenases of Desulfovibrio vulgaris Hildenborough

An original gas chromatographic-mass spectrometric technique is described for studying simultaneous dihydrogen-deuteron exchange and para-ortho H 2 conversion catalyzed by different Desulfovibrio hydrogenases. Para and orthohydrogens are separated on an alumina column at the temperature of liquid nitrogen, but if both HD and ortho H 2 are present, their retention times are too close to each other for total separation and only one peak is observed with a thermal conductivity detector. In order to resolve the peaks from one another, a fraction of the gas released from the gas chromatograph column is admitted to the ion source of a mass spectrometer, where the gases are separated according to their respective masses. Because of a peak-jumping system, the different components involved in the exchange and in the conversion reactions can be scanned so that the spectra corresponding to mass m e 2 (para and ortho H 2), m e 3 (HD), and m e 4 (D 2) can be obtained simultaneously. This technique has been employed to resolve a controversial problem concerning the occurrence or lack of any para-orthohydrogen conversion in heavy water. Actually both exchange and conversion were demonstrated to occur with a (NiFe) hydrogenase, whereas with a (NiFeSe) hydrogenase, which had an exchange activity equivalent to that of the former, practically no para-ortho conversion could be observed in D 2O. These findings are related to the constitutional and catalytic properties of the hydrogenases belonging to the different classes.

In vivo conversion of γ-aminobutyric acid and 1,4-butanediol to γ-hydroxybutyric acid in rat brain : Studies using stable isotopes

The formation of 4-[1,4- 13C]hydroxybutyric acid ([ 13C]γ-hydroxybutyric acid; [ 13C]GHB) in rat brain was studied following intracerebroventricular (i.c.v.) administration of either 4-[1,4- 13C]aminobutyric acid ([ 13C]GABA or 1,4-[1,4- 13C]butanediol ([ 13C]1,4-BD) to awake, freely moving animals. GHB and [ 13C]GHB were measured with a gas Chromatographic mass spectrometric (GC/MS) technique designed to detect the lactone derivative of GHB with the acid or lactone being determined by conditions of tissue extraction. [ 13C]GHB was detected following i.c.v. administration of [ 13C]GABA with a turnover rate of 2.04nmol/g tissue/hr and [ 13C]1,4-BD with a turnover rate of 1.4 nmol/g/hr. The formation of [ 13C]GHB from [ 13C]GABA was blocked by an inhibitor of GABA-transaminase, but this drug had no effect on the formation of [ 13C]GHB from [ 13C]1,4-BD. The latter pathway was also unaffected by alcohol dehydrogenase inhibitors, compounds which block this pathway in the periphery. Further, in the course of these experiments, naturally occurring endogenous γ-butyrolactone (GBL) was detected in rat brain in a concentration of 200 pmol/g tissue weight, but lactonization in vivo of [ 13C]GHB formed from either labeled GABA or 1,4-BD was not demonstrated. These data confirm two separate pathways of synthesis for GHB in brain, demonstrate the presence of GBL in brain, and illustrate the utility of a new GC/MS technique for analysis of GHB and for GBL which does not involve extensive derivitization.

The effect of concussive head injury on central cholinergic neurons

This study examined the effect of fluid percussion head injury on the activity of cholinergic neurons in specific brain areas of the rat 12 min, 4 h and 24 h following injury. Acetylcholine (ACh) turnover, used as an index of cholinergic neuronal activity, was determined using a gas chromatographic-mass spectrometric technique. The most striking changes in cholinergic activity were observed in the dorsal pontine tegmentum, where concussive head injury produced an increase in ACh turnover 12 min and 4 h following injury. This area has been previously associated with behavioral changes observed following concussive injury. ACh turnover in the thalamus, a region to which pontine cholinergic neurons project, also tended to increase 4 h following injury. On the other hand, ACh turnover tended to decrease in the amygdala 4 h following injury. Although there were no significant changes in hippocampal ACh content or turnover following injury, ACh content did tend to increase in that brain region 12 min following injury. There were no significant effects of injury on cholinergic neurons in the cingulate/frontal cortex. These changes in cholinergic neuronal activity may contribute to the neurological deficits following concussive injury. In particular, activation of cholinergic neurons in the pontine region may contribute to components of behavioral suppression associated with reversible traumatic unconsciousness. More generalized changes in cholinergic function may lead to the production of more chronic deficits.

Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry.

To determine if levels of serum total homocysteine are elevated in patients with either cobalamin or folate deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure total homocysteine in the serum of 78 patients with clinically confirmed cobalamin deficiency and 19 patients with clinically confirmed folate deficiency. Values ranged from 11 to 476 mumol/liter in the cobalamin-deficient patients and 77 of the 78 patients had values above the normal range of 7-22 mumol/liter as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum total homocysteine was positively correlated with serum folate, mean corpuscular volume, serum lactate dehydrogenase, serum methylmalonic acid, and the degree of neurologic involvement, and inversely correlated with platelets and hematocrit. In the folate-deficient patients, values for serum total homocysteine ranged from 17 to 185 mumol/liter and 18 of the 19 patients had values above the normal range. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of total homocysteine while they were free of hematologic and neurologic abnormalities. The measurement of serum total homocysteine will help define the incidence of cobalamin deficiency and folate deficiency in various patient populations.

5-Hydroxytryptophol and 5-hydroxyindoleacetic acid levels in rat brain: Effects of various drugs affecting serotonergic transmitter mechanisms

The effect of various pharmacological treatments on the levels of free and total 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain were investigated using a gas chromatographic-mass spectrometric technique. The disappearance of 5-HTOL following monoamine oxidase (MAO) inhibition induced by pargyline was more rapid (t1/2 10-15 min) than that of 5-HIAA (t1/2 30-40 min) in all regions investigated, indicating a rapid turnover of 5-HTOL. The selective MAO-A inhibitor chlorgyline produced a more pronounced reduction of 5-HTOL than of 5-HIAA, while the MAO-B inhibitor deprenyl was without effect on both serotonin metabolites. The MAO-A inhibitor amiflamine which is selective for serotonin neurons, was also more effective in reducing free 5-HTOL levels than of 5-HIAA levels, suggesting that the formation of 5-HTOL is closely associated with serotonin neurons. Neonatal treatment with the serotonin neurotoxin 5, 7-dihydroxytryptamine (5,7-HT) led to a more pronounced reduction of 5-HTOL levels in cerebral cortex than that of 5-HIAA levels, while the increase of 5-HTOL levels in pons-medulla was more marked than of 5-HIAA levels. Probenecid treatment increased several fold both conjugated 5-HTOL and 5-HIAA levels in brain tissue. An increase was also noted for free 5-HTOL, although of less magnitude. Treatment with the serotonin receptor active agents methiothepin and d-lysergic acid diethylamide produced similar alterations of free 5-HTOL and 5-HIAA. The present results have demonstrated that free 5-HTOL has a rapid turnover in rat brain and that free 5-HTOL levels may serve as a useful index for serotonin turnover.

Determination by Gas Chromatography-Mass Spectrometry of [15N5]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue

In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [(15)N(5)]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium ((2)H)-labeled internal standards. Incorporation levels of the [(15)N(5)]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5'-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-(14)C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-(14)C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of (14)C incorporation into cytokinins declined.

Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatography-mass spectrometry.

To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.

Portal venous bile acids in cholesterol gallstone disease: effect of treatment with chenodeoxycholic and cholic acids.

We determined the serum concentrations of cholic, chenodeoxycholic and deoxycholic acids in portal and peripheral venous blood in 9 gallstone-free patients and 39 patients with cholesterol gallstones during standardized cholecystectomy. An accurate and specific gas chromatographic-mass spectrometric technique was used. The portal venous concentration of total bile acids was similar in gallstone-free and untreated gallstone patients (n = 20); there was no evidence of a reduced hepatic uptake of bile acids in the latter. Treatment with cholic acid (n = 10) was associated with a 70% increase in cholic acid and normal concentration of total bile acids. In chenodeoxycholic acid-treated patients (n = 9), the portal venous concentration of this bile acid was increased 3-fold; total bile acids were increased about 60%. The estimated hepatic uptake of cholic acid was slightly decreased during chenodeoxycholic acid treatment. The results indicate that neither bile acid inflow to the liver nor hepatic bile acid uptake is reduced in fasting patients with cholesterol gallstones, and treatment with chenodeoxycholic acid increases fasting inflow of bile acids to the liver. The latter may contribute to unsaturation of fasting hepatic bile during treatment with chenodeoxycholic acid.

Use of chemical ionization in gas chromatographic/mass spectrometric screening of human urine for disaccharides containing inositol.

Gas chromatography/mass spectrometry (GC/MS) in the positive chemical ionization (CI) mode was used to screen normal urine for inositol-containing disaccharides in the form of permethylated derivatives, after borodeuteride reduction of the reducing saccharides. Ammonia was the reactant gas. The results revealed the existence of deoxyhexosyl-inositol and hexosyl-inositol disaccharides, and of a new compound, N-acetylhexosaminyl-inositol disaccharide. Up to four isomers of deoxyhexosyl-inositol could be present in the same sample even though only one of them has so far been fully characterized in man. As regards the hexosyl-inositols, one to three isomers were present in the same sample and probably corresponded to the three isomers of galactosyl-inositol recently described in man. N-Acetylhexosaminyl-inositol (identified elsewhere by us as N-acetylgalactosaminyl-alpha (1-1)-myo-inositol) was seen in only a few samples. No relationship can be found between the excretion of all these inositol-containing disaccharides on the one hand, ABO(H) blood group and 'Secretor' status (Se or sese) of the donors on the other. The gas chromatographic CI mass spectrometric technique used here with various ammonia pressures can be applied to the screening of other biological fluids or tissues for inositol glycosides.